The applicant is a trained clinical endocrinologist with special interest in autoimmunity. His preliminary work in the molecular cloning of autoantibodies in Graves' ophthalmopathy has been a first step in a new approach toward understanding the pathogenesis of this disease. The primary goal of the project is to test the hypothesis that the human thyrotropin receptor (TSHR) is the elusive antigenic target of the immune response in Graves' ophthalmopathy. This hypothesis has been strengthened by the recent demonstration of TSHR, or variant TSHR, mRNA transcripts in retro-orbital tissues. The studies proposed are: 1)Generation of recombinant human TSHR -antigens:- (i) Wild-type TSHR and TSHR ectodomain: Stably transfected CHO cells expressing full- length wild-type and truncated TSHR ectodomain are available, but due to the low levels of expression, extensive effort involving cell culture and chromatographic procedures will be required to purify sufficient quantities of these proteins. (ii) Molecular cloning and expression of the TSHR 1.3 kb ectodomain splice variant (TSHRvl.3): The cDNA of the TSHRv1 .3 will be generated by PCR from a human thyroid cDNA library, cloned into a eukaryotic expression vector and stably transfected into CHO cells. Protein expression and partial purification will be as for the TSHR ectodomain. 2)Screening of TSHR antigens for recognition by autoantibodies in patients' sera: lmmunoblot analysis and ELISA: Comparison will be made of autoantibody recognition of TSHR 1.3 kb variant and TSHR ectodomain. Sera to be tested will be from Graves' patients with or without ophthalmopathy and other control individuals. 3)Molecular Cloning and expression of immunoglobulin genes from orbital plasma cells in Graves' ophthalmopathy:- (i) Construction of combinatorial IgG heavy (H) and light (L) chain cDNA libraries: cDNA will be prepared by reverse-transcription and PCR of mRNA from plasma cells in Graves' orbital tissue. Control libraries will be made from orbital tissue from patients without Graves' ophthalmopathy. Both bacteriophage lambda and filamentous phage ("phage display") libraries will be constructed. (ii) Combinatorial libraries: will be screened for human monoclonal autoantibodies (MAb) that recognize the recombinant TSHR antigens. The isolation of MAb specific for the TSHR, or variants, will be of potential value at least diagnostically.